Targeting HIV-1 with CRISPR/Cas9 delivered by retargeted adenoviruses effectively suppresses viral replication

Integrated, intact, latent HIV-1 viruses in infected cells are the main obstacle to curing HIV-1 infections. Targeted inactivation of HIV-1 proviruses with CRISPR/Cas9 is a promising strategy to eradicate HIV 1. In addition, CRISPR/Cas9 is able to target replicating HIV 1 and could be used as a therapy during productive infection. Here, we combine the CRISPR/Cas9 system with a novel adenovirus (Ad) targeted delivery technology to test it as a therapeutic approach to inhibit HIV-1. First, we selected six HIV-1-specific gRNAs targeting the HIV 1 LTRs and the gag gene and tested their efficacy in inhibiting HIV-1 virion production in an HEK 293T cell co-transfection screen. The gRNA-TAR showed the most robust and potent inhibition of HIV-1 by >99% alone or in combination with the gRNA-p24, which induced a ~1 kb deletion between both gRNA target sites in HIV-1 DNA. Delivery of this dual gRNA-TAR/p24 CRISPR/Cas9 system with CD3-CD28-IL2-retargeted Ads was highly effective, transducing 62.3 +/- 23.3% of cells and suppressing HIV-1 replication by 88.0 +/- 4.5% in primary CD4+ T cells from three independent donors. Our dual gRNA-TAR/p24-CRISPR/Cas9-Ad strategy represents a novel therapeutic approach to effectively inhibit HIV-1 in a highly HIV-1 and T cell-specific manner. ### Competing Interest Statement KJM has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV and Abbott; and the University of Zurich received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies that Dr. Metzner serves/served as principal investigator, and advisory board honoraria from Gilead Sciences. AP is a cofounder and shareholder of Vector BioPharma that commercializes the targeted and shielded adenovirus technology. The other authors declare no conflicts of interest.